The present invention relates to new methods of making biochips and the biochips resulting therefrom. In particular, the new method described herein provides for rapid, simple and cost effective construction of biochips by employing polyurethane-based hydrogels to immobilize biomolecular probes on the substrate. In particular, both organic solvent soluble biomolecules, like peptide nucleic acids (PNAs), and water soluble biomolecules, like DNA, RNA and other oligonucleotides, are easily and efficiently bound to the hydrophilic polymer either before or during polymerization thereof. In addition to the improved method of making the biochips described herein, the biochips themselves have improved characteristics including superior stability providing a much improved shelf-life and greater flexibility in use. For example, the biochips of the present invention are useful for gene discovery, gene characterization, functional gene studies, screening for biological activity and related studies.
Agents that selectively bind to DNA or RNA are of significant interest to molecular biology and medicinal chemistry as they may be developed into gene-targeted drugs for diagnostic and therapeutic applications and may be used as tools for sequence-specific modification of DNA. Additionally, such reagents may be used as tools for determining gene sequences and for functional gene analyses. Peptide nucleic acids (PNAs) are a recently developed class of oligonucleotide mimics wherein the entire deoxyribose phosphate backbone has been replaced by a chemically different, structurally homomorphous backbone composed of (2-aminoethyl) glycine units. Despite this dramatic change in chemical makeup, PNAs recognize complimentary DNA and RNA by Watson-Crick base pairing. Furthermore, PNAs have been shown to have numerous advantages over DNA and RNA oligomers. For example, PNAs lack 3' to 5' polarity and thus can bind in either a parallel or an anti-parallel orientation to DNA or RNA. (Egholm, M. et al., Nature 365:566, 1993). Another advantage to PNAs is that their selective hybridization to DNA is less tolerant of base pair mismatches than DNA--DNA hybridization. Thus, PNAs are becoming a very useful tool in the study of genes.
Until recently, the processes of gene discovery, characterization and functional gene analysis have been difficult and costly and have required tremendous amounts of time. However, within about the last ten years, methods of isolating arrays of biomolecules on various supports, referred to as biochips, have been developed and have been employed in DNA synthesis, sequencing, mutation studies, gene expression analysis and gene discovery. Generally the biochips are micro matrices (i.e., micro arrays) of molecules bound to a substrate, either directly or through a linking group or, more recently, by way of a gel layer. Most biochips are designed to facilitate synthesis of biomolecules at known locations on a substrate. For example, one such biochip employs light and a series of photo-lithographic masks to activate specific sites on a substrate, such as glass, in order to selectively bind nucleic acids thereto and, subsequently, to attach additional nucleic acids to form known oligonucleotides at the desired locations. This process of using light and photolithographic masks to activate specific sites on a substrate is similar to the processes used in production of the microelectronic semi-conductor chip. Unfortunately, these first generation biochips are very expensive to produce, requiring capital investments in the neighborhood of 2 to 4 million dollars. Furthermore, this synthesis method of forming oligonucleotides in a single layer on a substrate results in a low sensitivity biochip requiring an expensive laser confocal fluorescence microscope for adequate detection of DNA specifically hybridized to the chip. See, for example, U.S. Pat. No. 5,744,305, issued to Fodor, et al. on Apr. 28, 1998 (hereinafter, the '305 patent), which provides an example of the use of photolabile protecting groups and photolithography to create arrays of materials attached to a substrate. The '305 patent, which is hereby incorporated by reference, in its entirety, describes a synthetic strategy for the creation of large scale chemical diversity wherein a substrate, such as glass, is derivatized, either directly or by addition of a linker molecule, to include an amino group blocked with a photolabile protecting group. Masking techniques are then employed to permit selective deprotection of the photolabile groups within a specified, known location on the substrate. The deprotected region is then reacted with a "building block" molecule, for example an oligonucleotide, also containing a photolabile protecting group, such that the building block molecule is covalently bound to the active group at the surface of the substrate (or linker). This process is then repeated using the masks to direct synthesis of polymers, for example peptides, of specific, known sequences at specific, predefined locations on the substrate.
The synthetic strategies described in the '305 patent contemplate providing from about 10 up to about 10.sup.8 different sequences on a single substrate. Additionally, it is stated that the predefined regions on the substrate, wherein individual polymers are to be synthesized, are from about 10.sup.-10 cm.sup.2 to about 1 cm.sup.2. While the examples presented in the '305 patent primarily involve synthesis of peptides or nucleotides, it is stated that the same techniques may also be employed in the synthesis of other polymers. Similarly, various linker groups, preferably inactive or inert, for linking the synthesized polymer to the substrate are discussed in the '305 patent, as are various protecting groups for protecting an active site on the monomers which protecting groups may be selectively removed for directed synthesis of the polymer. Also discussed in some detail in the '305 patent is a binary masking technique utilized in one embodiment for directed synthesis of the array. Unfortunately, the strategies described in the '305 patent suffer from many of the same disadvantages as other prior art methods and apparatus. The arrays are expensive to manufacture and use, require multiple steps and lengthy incubation/washing times during manufacture and, significantly, permit synthesis in only a single layer.
In view of the low sensitivity of these first generation biochips, second generation biochips have been developed.
One example of a second generation biochip is described in U.S. Pat. No. 5,736,257 (hereinafter the '257 patent), issued to Conrad, et al., on Apr. 7, 1998, and U.S. Pat. No. 5,847,019, issued to Conrad, et al. on Dec. 8, 1998 (hereinafter, the '019 patent), both of which patent are hereby incorporated by reference, in their entirety. The '257 and '019 patents describe a process of synthesizing a biochip comprising providing a substrate, such as glass, having surface hydroxyl groups; reacting the substrate surface hydroxyl groups with silanes to bind a molecular layer of vinyl groups upon the substrate; placing an acrylaimide compound on the molecular layer, which acrylamide compound can participate in a free radical polymerization reaction to make a polymerized network layer bound to the molecular layer; photo-activating the polymerized network layer to make a patterned photo-activated polymerized network; and placing upon the photo-activated polymerized network layer, for example by synthesis thereon, one or more similar or dissimilar biomolecules to bind to the patterned photo-activated polymerized network layer.
The biochips disclosed in the '257 and '019 patents are somewhat similar to the first generation biochips of Fodor, et al. in that they employ photolithographic techniques to direct binding (or synthesis) of biomolecules to an array. However, in contrast to the first generation biochips, the biochips of the '257 and '019 patents employ a polyacrylamide network on top of a molecular layer of vinyl groups, thereby giving a third dimension to the gel cells. Still, as will be readily appreciated by those of skill in the art, production of biochips in accordance with the disclosures of the '257 and '019 patents is not only expensive but also time consuming.
U.S. Pat. No. 5,552,270 (hereinafter the '270 patent), issued to Khrapko, et al., on Sep. 3, 1996, describes another second generation biochip. The '270 patent, hereby incorporated by reference in its entirety, describes a biochip comprising a solid support and a matrix, including an array of oligonucleotides at desired lengths, the matrix being attached to the support by means of a gel layer having a thickness of no more than 30 .mu.m. The gel layer described in the '270 patent preferably consists of a set of spaced "dots", according to the number of matrix elements. In contrast to the single layer formats described in the '257 and '305 patents, the gel layer of the '270 patent provides for a three-dimensional attachment of oligonucleotides to the substrate at a capacity therefore exceeding the capacity of the mono-molecular layer of the first generation biochips. This second generation biochip employs a polyacrylamide gel sandwiched between two glass slides that are spaced from one another with spacers no more than 30 .mu.m thick. Following polymerization of the polyacrylamide gel, one of the slides is removed and the gel-coated lower slide is dried and part of the gel is removed, for example, mechanically, so that the gel portions are separated by interstices, generally of not more than 30 .mu.m, that remain on the slide surface. In an alternative embodiment described in U.S. Pat. No. 5,770,721 (hereinafter the '721 patent), issued to Ershov, et al., on Jun. 23, 1998, the gel portions to be removed from the slide are removed using a laser. The '721 patent is also hereby incorporated by reference in its entirety for all purposes.
Even these second generation biochips, however, continue to have significant disadvantages. One disadvantage is that the polyacrylamide gel used to form the gel matrix is susceptible to evaporation of water, therefore, the chip must be covered with a non-volatile oil during storage, requiring additional washing with organic solvent, such as chloroform or ethanol, before use for hybridization experiments and reducing the shelf-life of the biochip. Another significant disadvantage to these second generation biochips is cost. Although they do provide increased detection sensitivity, as compared with the first generation biochips, the overall manufacturing cost of this three-dimensional polyacrylamide biochip is still very high due to the complex processes required to produce the chip. In particular, production of this biochip requires lengthy and cumbersome multi-step processing including; polymerization and binding to the surface of the glass substrate; mechanical or laser cutting to form micro-squares of gel matrix on the substrate; an activation step using hydrazines; followed by reaction with the oligonucleotides. Due to the inherent polymerization process of polyacrylamide gels, these steps must be performed independently. Thus, the total time required to produce a single biochip by this method is at least about 24 to 48 hours. Furthermore, after each step, thorough washings and/or other special cares must be taken before the next step may be begun. For example, the oligonucleotide derivatization step requires a long incubation period, such as twenty-four to forty-eight hours. Still another significant disadvantage to the second generation biochips lies in the fact that the reaction of the oligonucleotides with the hydrazine groups forms unstable morpholine derivatives resulting in a very short shelf half-life for the biochip of approximately thirty-six hours at room temperature. Thus, there is a significant need in the industry for a simple, cost effective, rapid method for constructing a reliable multi-functional biochip having a reasonably long shelf-life that may be used in gene discovery, gene characterization, functional gene analysis and related studies.